Serpinf2-Binding Molecules and Method of Use Thereof

ABSTRACT

Compositions and methods of using SerpinF2-binding molecules for preventing and/or reducing organ damage, functional disability or mortality in a patient at risk due to the activity of SerpinF2 and/or plasminogen activators on tissue injury. Also provided are compositions and methods of using SerpinF2-binding molecules for inhibiting hemorrhage, edema, and apoptosis. Methods for the preparation of medicaments for such methods of treatment and prevention are provided.

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/198,804, filed on Mar. 6, 2014, which is a continuation of PCTApplication No. PCT/US2012/053900, filed on Sep. 6, 2012, which claimspriority to U.S. Provisional Application No. 61/531,278, filed Sep. 6,2011, the entire contents of which are incorporated by referenceherewith.

ACKNOWLEDGEMENT OF FEDERAL RESEARCH SUPPORT

This invention was made, in part, with Government support under NationalInstitute of Health Grant Nos. HL092750 & NS073147. Accordingly, theUnited States Government has certain rights in this invention.

FIELD OF THE INVENTION

This invention relates generally to compositions and methods forpreventing or reducing morbidity, disability and death from cellulardamage, hemorrhage and organ swelling after tissue injury, due to theactivity of SerpinF2 in conditions typically associated with increasedlevels of plasminogen activators.

BACKGROUND

Tissue injury to the heart, brain, kidney and lungs may trigger thedeath of cells from toxicity, necrosis, apoptosis and other mechanisms.This triggers structural degradation of organ components, breakdown ofvascular barriers and cellular swelling. The result may be organ edema,hemorrhage and loss of function. For example, brain edema or swelling isa feared complication of trauma, injury or stroke that can cause deathor disability.¹ Brain swelling may also follow hemorrhage.² In the eye,macular edema may occur following central retinal vein occlusion.³Myocardial edema is an early marker of myocardial ischemia.^(4,5)Ischemia-reperfusion increases lung permeability and induces lung edemaas well.⁶ Ischemia and reperfusion in one organ may cause edema in thatorgan and, in addition cause swelling and dysfunction in others. Forexample, ischemia-reperfusion of the bowel may result in edema of thebowel as well as the kidney and lung.⁷ Similarly, ischemia in the livermay result in liver and kidney injury and edema.⁸ Ischemia andreperfusion lead to breakdown of the vascular barrier and edema of thepancreas.⁹ Ischemia and reperfusion leads to death and apoptosis ofendothelial cells.¹⁰ Microvascular injury occurs after ischemia andreperfusion.¹¹

Stroke is a worldwide public health issue that kills more than 5.7million people per year and is a leading cause of disability.¹² Strokeincreases the expression of matrix metalloproteinases, to promote thebreakdown of the blood brain barrier, to increase brain swelling oredema and to enhance the risk of hemorrhage. In patients with stroke,90% of the deaths within the first week are due to neurological causessuch as brain swelling and hemorrhage.¹⁵⁻¹⁷ Strokes with large amountsof cerebral edema are considered malignant or massive because they cancause increased intracranial pressure and loss of consciousness.Increased intracranial pressure resulting from edema and/or bleeding isassociated with a high mortality and may lead to herniation.^(18,19) Thefinding of significant brain swelling signifies a bad prognosis forpatients, while measurements of infarct size have not been considered tobe significant clinical predictors of disability.^(20,21)

Tissue plasminogen activator (TPA) catalyzes the production of the bloodclot-dissolving enzyme plasmin and is the only FDA-approved treatmentfor stroke. Unfortunately, the therapeutic benefit of TPA appears to belimited by its harmful or neurotoxic effects. TPA reduces disability inonly 11-13% of treated patients.²²⁻²⁴ TPA also significantly increasesthe risk of breakdown of the blood brain barrier resulting in brainhemorrhage which occurs in a dose-related fashion.²⁵ Administration ofTPA to patients after prolonged ischemia may increase mortality.²⁶

TPA is expressed by endothelial cells and by neurons and, thus ispresent both in the vascular space and the brain parenchyma.²⁷ Levels ofendogenous TPA rise in the brain in response to injury.²⁸⁻³⁰ In modelsof mechanical (non-thrombotic) occlusion of the middle cerebral artery(MCA), endogenous TPA increases neuronal cell death and pharmacologicadministration of TPA further enhances brain injury.³¹⁻³³ Neuronaldamage after a cerebral infarct is thought to be mediated in part byexcitotoxins.²⁷ It has been shown that TPA enhances excitotoxic braininjury³⁴ through a plasminogen-dependent mechanism and that SerpinF2(also known as α2-antiplasmin), the serine protease inhibitor (serpin)of plasmin is protective.³⁵⁻³⁷ Taken together, these data in mechanicalocclusion models indicate that TPA exerts neurotoxic effects on thebrain through its production of plasmin and, inhibition of plasminactivity by SerpinF2 reduces neurotoxicity. Yet paradoxically, for humanischemic stroke, which is typically caused by thrombotic(non-mechanical) occlusion, SerpinF2 is a risk factor which suggeststhat it may exerts negative effects.^(38,39)

In addition to the brain, endogenous or administered TPA has harmfuleffects after ischemia in other tissues throughout the body. Afterischemia in the kidneys, TPA increases tissue damage.⁴⁰ In a similarmanner, after ischemia in the lungs, TPA enhances lung injury anddiminishes lung function.⁴¹ TPA has also been shown to increase myocytetissue damage after cardiac ischemia.⁴² Similar to its harmful effectson neurons, TPA also enhances retinal cell damage induced byexcitotoxins in the eye.⁴³

U.S. Pat. No. 6,946,438 to Nagai et al. provides the use of compounds,such as plasmin, mini-plasmin and micro-plasmin, that reduceα2-antiplasmin (SerpinF2) concentration or activity in vivo, for thetreatment of focal cerebral ischemia infarction induced in animals bymechanical occlusion. However, mechanical occlusion does not simulatehuman ischemic stroke, which is predominantly caused by thrombosis orembolism of a clot (thromboembolism). The presence of a thrombus isassociated with fibrin products and activation of platelets and thecoagulation system, which may affect the ischemic microvasculature,trigger downstream thrombosis and have neurotoxic effects on neurons andother cells.⁴⁴. It has been found that mechanical occlusion induces adifferent pattern of cellular injury associated with TPA than thatcaused by thrombotic occlusion.^(30,44-46) For example, Nagai et al.found contradictory results for PAI-1 transgenic mice in a mechanicalocclusion and in a thrombotic stroke occlusion model.³⁰ Since studies bythese same authors suggest that compounds that reduce focal ischemiainfarction induced by mechanical occlusion can have opposite effects onischemic stroke induced by thrombosis, it is not predictable whethercompounds described by Nagai et al. in U.S. Pat. No. 6,946,438 willreduce neuronal cell death triggered by thrombotic occlusion. Inaddition in U.S. Pat. No. 6,946,438, Nagai et al. do not teach whethersuch compounds may prevent disability, brain swelling, hemorrhage ordeath after ischemic stroke. Since mechanical occlusion does notsimulate thrombotic stroke and does not adequately predict the value ofpotential therapies, there is a need to develop a composition and methodof preventing or reducing cellular damage, swelling, edema andhermorrhage in ischemic conditions caused by thrombosis, such asthromboembolic stroke.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions for inhibitinghemorrhage, organ edema, prolonged ischemia, breakdown of themicrovascular barrier, apoptosis or TPA toxicity in a patient,comprising administering to the patient an effective amount of aSerpinF2-binding molecule that reduces SerpinF2 activity orconcentration. The present methods of inhibition include methods for theprevention and treatment of the conditions described herein.

The invention also provides methods for the manufacture of a medicamentfor the treatment of all of the conditions described herein. The presentinvention provides that in various embodiments the SerpinF2-bindingmolecule is a SerpinF2 inhibitor selected from an antibody, a peptide, aDNA aptamer or a small molecule. In certain embodiments, the SerpinF2inhibitor is an antibody. In certain embodiments, the SerpinF2 inhibitoris administered in a dose range from 28-91 nanomoles/kg.

In particular, the present invention provides a method of inhibitingfunctional disability or death from hemorrhage or edema in a patient inneed thereof comprising administering to the patient an effective amountof a SerpinF2-binding molecule that reduces SerpinF2 activity orconcentration, thereby inhibiting disability or death from hemorrhage oredema in the patient. In certain embodiments, the hemorrhage or edema isneurologic, cardiac, hepatic, pancreatic, respiratory or renal.

The present invention provides a method of inhibiting disability ordeath from tissue plasminogen activator (TPA) toxicity in a patient inneed thereof comprising administering to said patient an effectiveamount of a SerpinF2-binding molecule that reduces SerpinF2 activity orconcentration, thereby inhibiting disability or death from TPA toxicity.In certain embodiments, the TPA toxicity causes hemorrhage, organ edema,or apoptosis. In certain embodiments, the invention comprises theearlier step of determining that the patient is at risk for TPA induceddamage. In certain embodiments, the TPA toxicity is due to ischemia ortrauma. The invention provides that the TPA toxicity can causeneurologic, cardiac, hepatic, pancreatic, respiratory or renal damage.In certain embodiments, TPA toxicity is assessed by determining that TPAhas been previously administered to the patient within 48 hours. Incertain embodiments, a plasminogen activator or serine protease enzymehas been previously administered to the patient within 48 hours.

The invention provides a method of preventing apoptosis in a patient inneed thereof comprising, administering to the patient an effectiveamount of a SerpinF2-binding molecule that diminishes SerpinF2 activityor concentration, thereby preventing apoptosis in the patient. Incertain embodiments, the apoptosis occurs in neurologic, cardiac,hepatic, pancreatic, lung or renal cells.

The invention provides a method of inhibiting prolonged ischemia in apatient in need thereof comprising administering to said patient aneffective amount of a SerpinF2-binding molecule that reduces SerpinF2concentration or activity in said patient so as to inhibit the prolongedischemia. In certain embodiments, the prolonged ischemia has beenpresent for at least forty (40) minutes. In certain embodiments, theprolonged ischemia occurs in neurologic, cardiac, hepatic, pancreatic,lung or renal tissues. In certain embodiments, the method comprises theearlier step of determining that the patient has neurologic symptomsindicative of neuronal damage. In certain embodiments, the neurologicsymptoms are classified as greater than or equal to Rankin 1 or NIHStroke Scale 4.

BRIEF DESCRIPTION OF THE DRAWINGS

Middle cerebral artery (MCA) thromboembolism reduces hemispheric bloodflow and causes neuronal cell death. (FIG. 1A) Hemispheric blood flowafter MCA thromboembolism (arrow) as measured by laser Doppler.Thromboembolus (arrow) in the MCA as viewed from the base of the brain(FIG. 1B).

SerpinF2 causes neuronal cell death and brain swelling. Mice treatedwith SerpinF2 (SF2) or nothing (control) experienced ischemia induced bythromboembolism (FIG. 2A). Neuronal cell death as assessed by TTCstaining in controls and SF2-treated mice. Neuronal cell death measuredas percent of hemispheric brain volume. (*p<0.01 vs. control, FIG. 2B).Brain swelling as percent of the brain hemisphere. (*p<0.05 vs. control,FIG. 2C).

Agents that inhibit or inactivate SerpinF2 (SF2-I) reduce mortality,neuronal injury, edema, hemorrhage and disability. FIG. 3A) SF2-I in theform of whole antibody (Ab) or Fab fragments prevent death by comparisonto control or TPA-treated mice. (***p<0.0005 vs TPA; p<0.005 vs.control). FIG. 3B) SF2-I reduce neuronal cell death (measured as percentof hemispheric volume; **p<0.001 vs control or TPA). FIG. 3C) SF2-Iprevent hemorrhage. (**p<0.01 vs control; p<0.05 vs. TPA). FIG. 3D)SF2-I prevent brain swelling or edema. (***p<0.001 vs control; p<0.05vs. TPA). FIG. 3E) SF2-I prevent behavioral disability by comparison tosham mice without strokes. Disability was measured by performance on aRotarod.

Agents that inhibit or inactivate SF2 (SF2-I) prevent breakdown of theblood brain barrier (BBB) (FIG. 4A), MMP-9 expression (FIG. 4B) andapoptosis as measured by TUNEL-staining (FIG. 4C) or caspase 3 cleavage(FIG. 4D). (**p<0.01, ***p<0.001 SF2-I vs. controls).

Neurotoxic effects of TPA on ischemic brains despite successful lysis.Mice were treated with standard dose TPA (10 mgs) or low dose TPA (2mgs) after 2.5 hrs. of ischemia induced by thromboembolism. FIG. 5A)Neuronal cell death measured as percent of hemispheric brain volume.FIG. 5B) Percent lysis or dissolution of the thromboembolus. FIG. 5C)Brain hemorrhage assessed as the percent of hemispheric brain volume(**p<0.01 vs. control).

Agents that inhibit or inactivate SF2 (SF2-I) abrogate the neurotoxiceffects of TPA to reduce neuronal cell death and hemorrhage. After 2.5hours of ischemia induced by thromboembolism, mice were treated withstandard (10 mg/kg) or low dose (2 mg/kg) TPA with or without aSerpinF2-inhibitor (SF2-I). FIG. 6A) Neuronal cell death measured aspercent of hemispheric brain volume. FIG. 6B) Brain hemorrhage assessedas the percent of hemispheric brain volume (**p<0.01 TPA alone vs.TPA+SF2-I).

Agents that inhibit or inactivate SF2 (SF2-I) abrogate the neurotoxiceffects of TPA to reduce neuronal cell death, hemorrhage and brainswelling in stroke survivors. After thromboembolic stroke, mice weretreated with TPA alone (10 mg/kg) or TPA (2 mg/kg) with anSF2-inhibitor. FIG. 7A) Neuronal cell death measured as percent ofhemispheric brain volume. FIG. 7B) Brain hemorrhage assessed as thepercent of hemispheric brain volume. FIG. 7C) Brain swelling as apercent of hemispheric volume. (*p<0.05, **p<0.01 or ***p<0.001 TPAalone vs. TPA+SF2-I).

Agents that inhibit or inactivate SF2 (SF2-I) abrogate the neurotoxiceffects of TPA to prevent breakdown of the blood brain barrier (BBB)(FIG. 8A), MMP-9 expression (FIG. 8B) and apoptosis measured byTUNEL-staining (FIG. 8C). (*p<0.05, **p<0.01, ***p<0.001 TPA vs.TPA+SF2-I).

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to thefollowing detailed description of the preferred embodiments of theinvention and the Examples included herein. However, before the presentcompounds, compositions, and methods are disclosed and described, it isto be understood that this invention is not limited to specificpolypeptides, specific nucleic acids, specific cell types, specific hostcells, specific conditions, or specific methods, etc., as such may, ofcourse, vary, and the numerous modifications and variations therein willbe apparent to those skilled in the art. It is also to be understoodthat the terminology used herein is for the purpose of describingspecific embodiments only and is not intended to be limiting.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the,” include plural forms unless thecontext clearly indicates otherwise. Thus, for example, reference to “anagent” includes one or more of such different agents, and reference to“the method” includes reference to equivalent steps and methods known tothose of ordinary skill in the art that could be modified or substitutedfor the methods described herein.

Unless otherwise defined, all technical and scientific terms used hereinhave the meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. The practice of the presentinvention employs, unless otherwise indicated, conventional techniquesof cell biology, molecular biology, genetics, chemistry, microbiology,recombinant DNA, and immunology. See, for example, Maniatis et al.(1982) Molecular Cloning, A Laboratory Manual, latest edition, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.; Sambrook et al.(1989) Molecular Cloning: A Laboratory Manual, latest edition (ColdSpring Harbor, N.Y.: Cold Spring Harbor Laboratory Press); Ausubel etal. (1992) Current Protocols in Molecular Biology, latest edition (NewYork: John Wiley & Sons); Guthrie & Fink (1991) Methods Enzymol.194:1-863; Cell Biology, A Laboratory Manual, ed. Celis, J. E., AcademicPress, NY; Histochemistry, Pearse, A. G. E., Vol. 1 (1980), Vol. 2(1985), and Vol. 3 (1990).

The present invention provides methods for inhibiting hemorrhage, organedema, prolonged ischemia, breakdown of the microvascular barrier,apoptosis or TPA toxicity in a patient, comprising administering to thepatient an effective amount of a SerpinF2-binding molecule that reducesSerpinF2 activity or concentration. The present methods of inhibitioninclude methods for the prevention and treatment of the conditionsdescribed herein.

The invention also provides methods for the manufacture of a medicamentfor the treatment of the conditions described herein. The presentinvention provides that in various embodiments the SerpinF2-bindingmolecule is a SerpinF2 inhibitor selected from an antibody, a peptide, aDNA aptamer or a small molecule. In certain embodiments, the SerpinF2inhibitor is an antibody. SerpinF2 inhibitors by directly binding toactive sites on SerpinF2, or indirectly by binding other regions ofSerpinF2 to sequester or otherwise reduce or diminish SerpinF2 activity,and thereby reduce the cellular damage associated with TPA toxicity. Incertain embodiments, the SerpinF2 inhibitor is administered in a doserange from 28-91 nanomoles/kg.

In particular, the present invention provides a method of inhibitingfunctional disability or death from hemorrhage or edema in a patient inneed thereof comprising administering to the patient an effective amountof a SerpinF2-binding molecule that reduces SerpinF2 activity orconcentration, thereby inhibiting disability or death from hemorrhage oredema in the patient. In certain embodiments, the hemorrhage or edema isspecific to any one or more of neurologic, cardiac, hepatic, pancreatic,respiratory or renal tissue.

The invention provides a method of preventing apoptosis in a patient inneed thereof comprising, administering to the patient an effectiveamount of a SerpinF2-binding molecule that diminishes SerpinF2 activityor concentration, thereby preventing apoptosis in the patient. Incertain embodiments, the apoptosis occurs in any one or more ofneurologic, cardiac, hepatic, pancreatic, lung or renal cells.

The present invention provides a method of inhibiting disability ordeath from tissue plasminogen activator (TPA) toxicity in a patient inneed thereof comprising administering to said patient an effectiveamount of a SerpinF2-binding molecule that reduces SerpinF2 activity orconcentration, thereby inhibiting disability or death from TPA toxicity.In certain embodiments, the TPA toxicity causes hemorrhage, organ edema,or apoptosis. In certain embodiments, the invention comprises theearlier step of determining that the patient is at risk for TPA induceddamage. In various embodiments, the TPA toxicity is, or is not, relatedto ischemia or trauma. The invention provides that the TPA toxicity cancause neurologic, cardiac, hepatic, pancreatic, respiratory or renaldamage. In certain embodiments, TPA toxicity is assessed by determiningthat TPA has been previously administered to the patient within 48hours. In certain embodiments, a plasminogen activator or serineprotease enzyme has been previously administered to the patient within48 hours.

The invention provides a method of inhibiting prolonged ischemia in apatient in need thereof comprising administering to said patient aneffective amount of a SerpinF2-binding molecule that reduces SerpinF2concentration or activity in said patient so as to inhibit the prolongedischemia. In certain embodiments, the prolonged ischemia has beenpresent for at least forty (40) minutes. In certain embodiments, theischemia has been prolonged for at least fifty (50) minutes, one (1)hour, two (2) hours, three (3) hours, four (4) hours, five (5) hours,and longer. In certain embodiments, the prolonged ischemia occurs in anyof neurologic, cardiac, hepatic, pancreatic, lung or renal tissues. Incertain embodiments, the method comprises the earlier step ofdetermining that the patient has neurologic symptoms indicative ofneuronal damage. In certain embodiments, the neurologic symptoms areclassified as greater than or equal to Rankin 1 or NIH Stroke Scale 4.Therefore, in certain embodiments, the invention also prolongs the timewindow for effective treatment in a patient with ischemia.

In certain embodiments, the hemorrhage, organ edema, prolonged ischemia,breakdown of the microvascular barrier, apoptosis or TPA toxicityresults from ischemia. In certain embodiments, the invention comprisesthe earlier step of determining that the ischemia is due to a thromboticischemic stroke. In certain embodiments, the invention further comprisesthe earlier step of determining that the ischemia is not due to amechanical occlusion. In certain embodiments, the hemorrhage, organedema, prolonged ischemia, breakdown of the microvascular barrier,apoptosis or TPA toxicity are not in brain tissues and result from acondition other than stroke.

The present invention also provides compositions and methods of usethereof, of decreasing neuronal damage, functional disability ormortality in a patient associated with a prolonged ischemia at risk forthe neurotoxicity induced by either an endogenous or externallyadministered plasminogen activator such as tissue plasminogen activator(TPA). The present disclosure describes for the first time thatSerpinF2-binding agents and/or molecules, e.g., SerpinF2 inhibitors, canbe used for reducing the cellular toxicity of tissue plasminogenactivator (TPA) in thromboembolic stroke or ischemic damage caused byblood clots in brain as well as in other organs.

Reducing cellular damage in ischemia can be performed on any tissues inneed, including without limitation tissues of the central or peripheralnervous system, hepatic/splenic/reticuolendothelial system, kidney andgenitourinary system, cardiovascular system, respiratory system,endocrine system, skin, gastrointestinal system, neurosensory systemmusculoskeletal system, and hematopoietic-lymphatic system.

As used herein, a SerpinF2-binding agent or molecule can include, amongother molecules, antibodies (polyclonal or monoclonal). The term“antibody” (Ab) or “monoclonal antibody” (MAb) is meant to includeintact molecules as well as antibody fragments (such as, for example,F_(v), F_(ab) and F_((ab′)2) fragments), single chain antigen-bindingproteins, “humanized” antibodies, and chimeric antibodies which arecapable of specifically binding to SerpinF2. F_(ab) and F_((ab′)2)fragments lack the F_(c) fragment of intact antibody, clear more rapidlyfrom the circulation, and may have less non-specific tissue binding ofan intact antibody.

U.S. Pat. No. 6,114,506 and pending U.S. Publication No. 20100086536 toReed et al. disclose certain other uses for SerpinF2 (aka,α2-antiplasmin) binding molecules, including but not limited to MAb49C9, 70B11, 77A3, and RWR, all of which molecules are hereinincorporated by reference. Further exemplary SerpinF2-binding moleculesinclude the following commercially available antibodies: monoclonalantibodies to MAP4H9 (Molecular Innovations), 27C9 (MolecularInnovations), 14AP (Fitzgerald Industries), MPW14AP (antibodies-onlineGmbH), 3617 (American Diagnostics), goat polyclonal antibody to SerpinF2(Biopool), and other anti-human polyclonal and monoclonal antibodies toSerpinF2 available from Genetex, Thermo Scientific Pierce ProteinResearch Products. The invention also contemplates the use of humanizedand human antibodies constructed through molecular biology techniques.

The phrases “SerpinF2-binding” and “specifically binding” refer to abinding reaction that is determinative of the presence of thepolypeptide in a heterogeneous population of polypeptides and otherbiologics. Thus, under designated immunoassay conditions, the specifiedantibodies (or other binding agent) bound to a particular polypeptide donot bind in a significant amount to other polypeptides present in thesample. Selective binding of an antibody under such conditions mayrequire an antibody that is selected for its specificity for aparticular polypeptide. A variety of immunoassay formats may be used toselect antibodies that selectively bind with a particular polypeptide.For example, solid-phase ELISA immunoassays are routinely used to selectantibodies selectively immunoreactive with a polypeptide. See Harlow andLane, “Antibodies, A Laboratory Manual,” latest edition, Cold SpringHarbor Publications, New York, (1988), for a description of immunoassayformats and conditions that could be used to determine selectivebinding.

The antibodies of the present invention may be prepared by any of avariety of methods. For example, cells expressing SerpinF2 (orfractions, lysates, etc. thereof) can be administered to an animal inorder to induce the production of sera containing polyclonal antibodiesthat are capable of binding SerpinF2. In a preferred method, apreparation of SerpinF2 antibody of the present invention is preparedand purified to render it substantially free of natural contaminants.Such a preparation is then introduced into an animal in order to producepolyclonal antisera of greater specific activity.

The antibodies of the present invention may also be prepared using phagedisplay technology. Methods of preparing antibodies using phage displayare known in the art. See, for example, U.S. Pat. No. 5,565,332;Clarkson et al., 1991, Nature 352:624-628; Huse, 1989, Science246:1275-1281; Kang, 1993, Proc. Natl. Acad. Sci. USA 88:11120-11123;Marks, 1991, J. Mol. Biol. 222:581-597; and McCafferty et al., 1990,Nature 348:552-554.

In some instances, it is desirable to prepare monoclonal antibodies(SerpinF2-binding molecules) from various hosts. A description oftechniques for preparing such monoclonal antibodies may be found inStites et al., eds., “Basic and Clinical Immunology,” (Lange MedicalPublications, Los Altos, Calif., Fourth Edition) and references citedtherein, and in Harlow and Lane “Antibodies, A Laboratory Manual” ColdSpring Harbor Publications, New York, 1988. For example, monoclonalantibodies can be prepared using hybridoma technology. In general, suchprocedures involve immunizing an animal (preferably a mouse) with theantigen or with a cell which expresses the antigen. A preferred antigenis purified SerpinF2 or a fragment thereof. Suitable cells can berecognized by their capacity to secrete anti-SerpinF2 antibody. Suchcells may be cultured in any suitable tissue culture medium; however, itis preferable to culture cells in Earle's modified Eagle's mediumsupplemented with 10% fetal bovine serum (inactivated at about 56° C.),and supplemented with about 10 ug/l of nonessential amino acids, about1,000 U/ml of penicillin, and about 100 ug/ml of streptomycin. Thesplenocytes of such mice are extracted and fused with a suitable myelomacell line. The method of somatic cell fusion is described in Galfre, G.and Milstein, C., Meth. Enzymol. 73:3-46 (1981). After fusion, theresulting hybridoma cells are selectively maintained in HAT medium, andthen cloned by limiting dilution as described by Wands et al., 1981,Gastroenterology 80:225-232. The hybridoma cells obtained through such aselection are then assayed to identify clones which secrete antibodiescapable of binding SerpinF2.

Alternatively, additional antibodies capable of binding to the SerpinF2antigen may be produced in a two-step procedure through the use ofanti-idiotypic antibodies. Such a method makes use of the fact thatantibodies are themselves antigens, and that, therefore, it is possibleto obtain an antibody which binds to a second antibody. In accordancewith this method, SerpinF2-specific antibodies are used to immunize ananimal, preferably a mouse. The splenocytes of such an animal are thenused to produce hybridoma cells, and the hybridoma cells are screened toidentify clones which produce an antibody whose ability to bind to theSerpinF2-specific antibody can be blocked by the SerpinF2 antigen. Suchantibodies comprise anti-idiotypic antibodies to the SerpinF2-specificantibody and can be used to immunize an animal to induce formation offurther SerpinF2-specific antibodies.

It will be appreciated that Fab and F_((ab′)2) and other fragments ofthe antibodies of the present invention may be used according to themethods disclosed herein. Such fragments are typically produced byproteolytic cleavage, using enzymes such as papain (to produce F_(ab)fragments) or pepsin (to produce F_((ab′)2) fragments). Alternatively,SerpinF2-binding fragments can be produced through the application ofrecombinant DNA technology, through synthetic chemistry, orbiotinylation.

Also intended within the scope of the present invention are humanized orchimeric antibodies, produced using genetic constructs derived fromhybridoma cells producing the MAbs described above. Humanized antibodiesare antibodies in which the framework or other regions of the murine Abis replaced with the homologous regions of a nonmurine antibody.Chimeric antibodies are antibodies in which the murine constant regionhas been replaced with a non-murine constant region. Methods forproduction of chimeric antibodies are known in the art. See, for review:Morrison, Science, 229:1202-1207 (1985); Oi et al., BioTechniques 4:214(1986); see also, Cabilly et al., U.S. Pat. No. 4,816,567 (Mar. 28,1989); Taniguchi et al., EP171496 (Feb. 19, 1986); Morrison et al.,EP173494 (Mar. 5, 1986); Neuberger et al., WO8601533 (Mar. 13, 1986);Robinson et al., WO 8702671 (May 7, 1987); Boulianne et al., Nature312:643-646 (1984); and Neuberger et al., Nature 314:268-270 (1985).Methods for production of humanized antibodies are known in the art.See, for example, U.S. Pat. No. 5,585,089; Jones et al., Nature321:522-525 (1986); and Kettleborough et al., Protein Engineering4:773-783 (1991).

Also provided in the present invention are antibodies capable of bindingto both (1) human and nonhuman circulating SerpinF2 and (2) human andnonhuman fibrin crosslinked SerpinF2. Such antibodies are well known inthe art. See, for example, U.S. Pat. Nos. 4,946,778; 5,260,203;5,091,513; and U.S. Pat. No. 5,455,030, all of which are hereinincorporated by reference. Also intended within the scope of the presentinvention are variants of the antibodies described above.

Also provided in the present invention are SerpinF2-binding agents ormolecules which are specifically not antibodies or fragments thereof.Screening for such SerpinF2-binding agents or molecules is routine inthe art. Particular known compounds of interest or libraries ofcompounds generated through combinatorial chemistry techniques, forexample, can be screened for the desired binding and conversionactivity. Furthermore, phage display technology can be used to identifypeptides, for example, for the desired binding and conversion activity.In general, phage display describes a selection technique in which alibrary of variants of a peptide or protein is expressed on the outsideof a phage virion, while the genetic material encoding each variantresides on the inside (Sidhu et al., 2003, Chembiochem. 4:14; Ferrer etal., 1999, J. Pept. Res.: 54, 32; BouHamdan et al., 1998, J. Biol. Chem.273: 8009). This creates a physical linkage between each variant proteinsequence and the DNA encoding it, which allows rapid partitioning basedon binding affinity to a given target molecule by an in vitro selectionprocess called panning (Whaley et al., 2000, Nature, 405, 665). In itssimplest form, panning is carried out by incubating a library ofphage-displayed peptides with a plate (or bead) coated with the target,washing away the unbound phage, and eluting the specifically boundphage. The eluted phage is then amplified and taken through additionalbinding/amplification cycles to enrich the pool in favor of bindingsequences. After 3-4 rounds, individual clones are characterized by DNAsequencing and ELISA. Many variations of the phage display technologyare known to those of skill in the art which can be adapted for purposesof the present invention.

In one embodiment, a phage display peptide library is used such asprovided by New England Biolabs (Mass, Mass.). The pre-made randompeptide libraries, Ph.D. libraries, have been used for myriad similarapplications, including epitope mapping, identification ofprotein-protein contacts (Rozinov and Nolan, 1998, Chem. Biol. 5:713-28)and enzyme inhibitors (Rodi et al., 1999, J. Mol. Biol. 285:197-203).

As used herein, the term “patient” is intended to be human or nonhuman.Preferably, the patient is human. As used herein the term“administering” refers to various means of introducing a compositioninto a cell or into a patient. These means are well known in the art andmay include, for example, injection or infusion for parenteral delivery;tablets, pills, capsules, or other solids for oral administration; nasalsolutions or sprays; aerosols; inhalants; topical formulations;liposomal forms; and the like. As used herein, the terms “effectiveamount” and “therapeutic amount” refer to an amount that will result inthe desired result and may readily be determined by one of ordinaryskill in the art depending upon the specific activity of the chosenSerpinF2 inhibitor and the condition of the patient. In certainembodiments, an effective or therapeutic amount of a SerpinF2 inhibitoris in a dose range of 28-91 nanomole/kg, 4.2-13.65 mg/kg, or 0.5-1.0moles inhibitor to mole of SerpinF2.

The compositions of the present invention may be formulated for variousmeans of administration. As used herein, the term “route” ofadministration is intended to include, but is not limited tosubcutaneous injection, intravenous injection, intraocular injection,intradermal injection, intramuscular injection, intraperitonealinjection, intratracheal administration, epidural administration,inhalation, intranasal administration, oral administration, sublingualadministration, buccal administration, rectal administration, vaginaladministration, and topical administration. The preparation of anaqueous composition that contains a peptide, antibody or antibodyfragment, antisense nucleic acid, receptor decoy, ribozyme, sensepolynucleotide, double stranded RNA, RNAi, aptamer, or small moleculeagonist, as an active ingredient will be known to those of skill in theart in light of the present disclosure. Typically, such compositions canbe prepared as injectables, either as liquid solutions or suspensions;solid forms suitable for using to prepare solutions or suspensions uponthe addition of a liquid prior to injection can also be prepared; andthe preparations can also be emulsified.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form should be sterile and fluid to theextent that syringability exists. It should be stable under theconditions of manufacture and storage and should be preserved againstthe contaminating action of microorganisms, such as bacteria and fungi.

The compositions of the present invention can be formulated into asterile aqueous composition in a neutral or salt form. Solutions as freebase or pharmacologically acceptable salts can be prepared in watersuitably mixed with a surfactant, such as hydroxypropylcellulose.Pharmaceutically acceptable salts, include the acid addition salts(formed with the free amino groups of the protein), and those that areformed with inorganic acids such as, for example, hydrochloric orphosphoric acids, or such organic acids as acetic, trifluoroacetic,oxalic, tartaric, mandelic, and the like. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as, forexample, sodium, potassium, ammonium, calcium, or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine, histidine,procaine, and the like.

Suitable carriers include solvents and dispersion media containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, or sodium chloride. Theproper fluidity can be maintained, for example, by the use of a coating,such as lecithin, by the maintenance of the required particle size inthe case of dispersion and/or by the use of surfactants.

Under ordinary conditions of storage and use, all such preparationsshould contain a preservative to prevent the growth of microorganisms.The prevention of the action of microorganisms can be brought about byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. Prolongedabsorption of the injectable compositions can be brought about by theuse in the compositions of agents delaying absorption, for example,aluminum monostearate, and gelatin.

Prior to or upon formulation, the compositions of the present inventionshould be extensively dialyzed to remove undesired small molecularweight molecules, and/or lyophilized for more ready formulation into adesired vehicle, where appropriate. Sterile injectable solutions areprepared by incorporating the active agents in the required amount inthe appropriate solvent with various of the other ingredients enumeratedabove, as desired, followed by filter sterilization. Generally,dispersions are prepared by incorporating the various sterilized activeingredients into a sterile vehicle that contains the basic dispersionmedium and the required other ingredients from those enumerated above.

In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum-drying andfreeze-drying techniques that yield a powder of the active ingredient,plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Suitable pharmaceutical compositions in accordance with the inventionwill generally include an amount of the active ingredient admixed withan acceptable pharmaceutical diluent or excipient, such as a sterileaqueous solution, to give a range of final concentrations, depending onthe intended use. The techniques of preparation are generally well knownin the art as exemplified by Remington's Pharmaceutical Sciences, 16thEd. Mack Publishing Company, 1980, incorporated herein by reference. Itshould be appreciated that for human administration, preparations shouldmeet sterility, pyrogenicity, and general safety and purity standards asrequired by FDA Office of Biological Standards.

Pharmaceutical compositions are provided that comprise an effectiveamount of a compound or molecule used in the disclosed methods forpreventing and/or reducing cellular injury, neuronal damage, swelling,functional disability, mortality, and cerebral hemorrhage in a patientat risk for the neurotoxicity induced by either an endogenous orexternally administered tissue plasminogen activator (TPA) associatedwith a prolonged ischemia, and/or associated with an activity ofSerpinF2. Pharmaceutical compositions are also provided that comprise aneffective amount of a compound or molecule used in the disclosed methodsfor decreasing neuronal damage, functional disability, mortality orhemorrhage by prolonging the time window for effective treatment in apatient with ischemia.

Prolonged ischemia, trauma or cause of brain injury is first manifestedby neurologic symptoms which may include muscle weakness, alteredspeech, altered consciousness, seizure or other impairment of normalneurologic function. A physician or other suitably trained healthcareprofessional makes the determination of prolonged ischemic condition,trauma or a diagnosis of developing stroke, after interviewing andexamining the patient. The diagnosis can be confirmed or refuted byarteriography, or CT , MRI or PET scanning or other imaging tests of thebrain which may disclose evidence of arterial obstruction, brainhypoperfusion, infarction, neuronal cell damage, edema, etc.Additionally, diagnostic tests (e.g., imaging, EEGs, blood tests, etc.)can be used to identify conditions in which SerpinF2 inhibitors would beinappropriate, such as in cases of significant intracranial hemorrhage,non-ischemic seizures, etc. The invention provides for theadministration of SerpinF2-binding agents after determination ordiagnosis of prolonged ischemia, trauma or other injury to the brainwhich has resulted in neurologic symptoms and disability. Suchdisability can be assessed with clinical scales such as the Rankinscale, NIH Stroke Scale, Glasgow scale, etc.

The invention also provides for methods of administration ofSerpinF2-binding agents prior to expected ischemia, trauma or injury,provided that the patient has been excluded from unacceptable bleedingrisk. In such instances ischemia may be induced by occlusion of thecerebral vessels such as during carotid endarterectomy, followingcerebral embolism complicating procedures on the heart or majorarteries, or post heart valve surgeries, etc. It is understood that suchdeterminations of the patients' ischemic condition involve physicaltransformations of matter and/or the use of medical equipment throughthe manipulation of the patient under examination and the performance ofdiagnostic tests.

It should also be understood that the foregoing relates to preferredembodiments of the present invention and that numerous changes may bemade therein without departing from the scope of the invention. Theinvention is further illustrated by the following examples, which arenot to be construed in any way as imposing limitations upon the scopethereof. On the contrary, it is to be clearly understood that resort maybe had to various other embodiments, modifications, and equivalentsthereof, which, after reading the description herein, may suggestthemselves to those skilled in the art without departing from the spiritof the present invention and/or the scope of the appended claims.

EXAMPLES Methods

MCA thromboembolism model.^(47,48) Normal C57BL/6J adult mice (29-35 g)were obtained from The Jackson Laboratory (Bar Harbor, Me.). Mice werehoused in micro-isolation cages on a constant 12-hour light/dark cyclewith controlled temperature and humidity, and given access to food andwater ad libitum. Experiments adhered to the guidelines set forth in theGuide for the Care and Use of Laboratory Animals (DHHS Publication No.(NIH) 85-23 Revised 1985) and were performed under protocols approved bythe Medical College of Georgia's and the University of Tennessee'sInstitutional Animal Care and Use Committee. Mice were mechanicallyventilated using 1.5 to 2% isoflurane and O2 during surgery as describedusing a TOPO Dual Mode Ventilator (Kent Scientific, Torrington,Conn.).⁴⁹ Body temperature was maintained at 37° C. with a warming pad.Cerebral blood flow was monitored by a laser Doppler monitor with afiber optic probe (ADInstruments PowerLab 2/26, blood flow meter ML191,OxyFlo Probe MSF100XP). The left common carotid artery was isolatedafter a neck incision, and the external carotid, thyroid, and occipitalarteries were ligated. Microvascular clips were temporarily placed onthe common carotid and internal carotid arteries. A small arteriotomywas made on the external carotid artery for retrograde insertion of thePE8 catheter containing the clot. Clots were made with pooled freshfrozen from normal mice mixed with ¹²⁵I-fibrinogen (˜400,000 cpm/20 ul,PerkinElmer NEX430110UC) and stained with Evans blue dye. The PE8 tubecontaining the clots was counted in a gamma-scintillation counter,inserted into the left external carotid artery, threaded into the ICA uptowards the origin of the MCA and the thrombus was embolized at a speedof 0.45 mL/min in a volume of 100 ul saline. A Geiger-Muller counter wasused to confirm appropriate embolization.

At the appropriate time post-embolism, TPA (Genentech, South SanFrancisco) was given by bolus (20% of dose) followed by infusion (80% ofdose) over 30 min in saline in 300 ul via the contralateral jugularvein. In other experiments TPA and/or a SerpinF2 inhibitor (4H9,Molecular Innovations, Novi, Mich.), or SerpinF2 or saline (control)were administered via the contralateral jugular vein. After 4 hrs. ofischemia the animal was euthanized, citrated blood was isolated bycardiac puncture and tissues were perfused as we have described.⁴⁹ Thebrain was sectioned coronally into 2 mm sections and photographed todigitally image gross hemorrhage. The slices were incubated in TTC toidentify viable tissue. In experiments investigating survival anddisability after stroke, animals were administered the indicated agentsat 30 min. or more after thromboembolism.

The hemispheric size, area of gross hemorrhage and area of neuronalinjury were digitally analyzed by a blinded observer using Image ProPlus 6.2 software and multiplied by the slice thickness to determinevolume (mm)³ using Swanson's method.⁵⁰ The mean volume was determinedfrom at least 8 different measurements per brain. The means of theaverage values for each group were compared by a one way ANOVA with aNeuman Keuls correction. The amount of lysis was determined bycomparison of the residual thrombus radioactivity in the brain to thatof the initial clot as we have described.⁵¹

Plasminogen and fibrinogen levels were measured in plasma in duplicateafter stroke as we have described.⁵² The means of the average values foreach group compared by a one way ANOVA with a Neuman Keuls correction.

Data Analysis

Statistical analyses were performed as described above and differencesbetween groups were considered to be significant if P<0.05. Data arereported as mean±SEM.

Results

Thromboembolism typically reduced hemispheric blood flow by ˜80% (FIG.1A). Thromboemboli were readily detected in the proximal MCA (FIG. 1B)and there was blanching of the affected cortex (not shown). There werelarge areas of neuronal cell death in the thromboembolic group micetreated with placebo (controls) but no neuronal cell death in the shamgroup of mice that received no thromboemboli. There was significantfibrinolysis of the thromboembolus in the controls (20.6±2.5%)consistent with previous reports of enhanced endogenous TPA activityfollowing stroke.^(53,54)

In contrast to previous studies,³⁶ administration of SerpinF2unexpectedly increased, rather than decreased, neuronal cell damage bycomparison to controls. (p<0.01; FIGS. 3A, B). Administration ofSerpinF2 significantly decreased lysis of the thromboembolus whencompared to control mice or mice receiving the SerpinF2 inhibitor(p<0.01). Administration of SerpinF2 also markedly increased swelling oredema in the ischemic hemisphere, another unexpected finding (FIG. 3C,p<0.05). There was no cerebral hemorrhage detected in any of the controlor SerpinF2-treated mice.

Treatment of mice with a SerpinF2 inhibitor markedly reduced mortalityrates by comparison to TPA-treated mice (p<0.0005, FIG. 2a ) or controlmice (p<0.005, FIG. 3A). This effect was independent of the molecularform of the SerpinF2 inhibitor as both whole monoclonal antibody and Fabfragments saved lives by comparison to TPA (p<0.001, FIG. 3A) and tocontrols (p<0.01, FIG. 3A). The survival effect was also dose-dependent:lower doses of the SerpinF2 inhibitor were less effective than higherdoses (p=0.05, not shown) but still reduced mortality by comparison tocontrol and TPA (p=0.01). Microscopic examination of the brains of micesurviving the initial stroke period (≥12 hrs.) showed that SerpinF2inactivation, whether in the form of a whole antibody or Fab, reducedneuronal cell death by comparison to control or TPA-treated mice (FIG.3B, p<0.001). Inactivation of SF2 prevented brain hemorrhage whencompared to control mice (FIG. 3C, p<0.01) or those receiving TPA (FIG.3C, p<0.05). Inactivation of SerpinF2 by whole antibody or Fab preventedbrain swelling by comparison to controls (FIG. 3D, p<0.001) andTPA-treated mice (FIG. 3D, p<0.05). To determine functional limitationafter stroke, behavioral tests were performed after a week of recovery.Survival was markedly limited in control or TPA-treated mice, thereforesham mice that underwent the surgical procedure, but had no stroke, wereused for comparison. By comparison to sham mice without stroke,treatment with an SF2-I prevented mice from disability as judged bytheir ability to maintain balance on a rotating cylinder (Rotarod), astandard behavioral test for mice after stroke (FIG. 3E).⁵⁵

Normally, occlusion of the middle cerebral artery is associated withpoor neurologic recovery, higher mortality and brain edema orswelling.⁵⁶ Brain swelling is attributed to breakdown of the blood brainbarrier which permits movement of fluids from the blood into the braintissue. Opening of the blood brain barrier is due in part to increasesin endogenous TPA activity in the perivascular tissue after stroke.⁵⁷Microscopic analyses of control brains showed that levels of albumin, ablood protein, were increased several fold on the side of the brainaffected by stroke by comparison to the side of the brain without stroke(p<0.005) or by comparison to shams (p<0.001, not shown). Albuminstaining was most intense in the perivascular area. By comparison tocontrols, inhibition of SF2 significantly reduced albumin stainingconsistent with decreased blood brain barrier breakdown (FIG. 4A;p<0.01). MMP-9 contributes to breakdown of the blood brain barrier.⁵⁸Levels of MMP-9 rise after stroke⁵⁸ and are associated with increasedrisk of hemorrhage in humans.⁵⁹ MMP-9 expression was often found in thearea of astrocytes foot processes typically associated with the bloodbrain barrier. By comparison to control mice, inhibition of SF2significantly reduced MMP-9 expression (FIG. 4B; p<0.001). Sinceinhibition of SF2 reduced neuronal cell death, MMP-9 expression andbreakdown of the blood brain barrier, it may also decrease brain celldeath associated with apoptosis. Consistent with this notion, thepercent of TUNEL-stained cells was significantly decreased in micetreated with the SF2-inactivator by comparison to controls (FIG. 4C,p<0.01). In addition, staining for cleaved caspase 3, a more specificindicator of apoptosis, was also reduced in the mice treated with anSF2-inactivator (FIG. 4D; p<0.001).

TPA is currently the only FDA-approved treatment for ischemic stroke.Administration of a standard dose of TPA for mice (10 mg/kg) after 2.5hrs. of ischemia, which simulates the typical treatment time of humanstroke, significantly increased neuronal cell death by comparison tocontrol (p<0.01, FIG. 5A), indicating that TPA enhanced neuronal injury.The standard dose of TPA also significantly increased dissolution of thethromboembolus (p<0.01, FIG. 5B) and caused a marked increase in brainhemorrhage (p<0.05, FIG. 5C). Administration of a lower dose of TPA (2mg/kg) after 2.5 hrs. of ischemia enhanced neuronal cell death whencompared to control mice (p<0.01, FIG. 5A). The lower dose of TPA didnot significantly increase the dissolution of the thrombus or increasehemorrhage (FIGS. 5B & C).

Previous studies had suggested that administration of SerpinF2 mayreduce TPA-induced neurotoxicity.³⁵ Surprisingly, however,administration of standard dose TPA (10 mg/kg) with a SerpinF2 inhibitormarkedly reduced neuronal damage by comparison to TPA alone (FIG. 6A,p<0.01). In a similar fashion, administration of low dose TPA with aSerpinF2 inhibitor significantly reduced neuronal damage when comparedto low dose TPA alone (p<0.01). Finally, administration of standard doseTPA with a SerpinF2 inhibitor markedly reduced the hemorrhage caused bystandard dose TPA alone (FIG. 6B, p<0.01).

Given that SerpinF2 appeared to enhance TPA's effect on neuronal celldeath, it was examined whether an SF2-I could reduce TPA associatedmortality after thromboembolic stroke. Mortality was 78% after TPAtreatment but 0% when mice were treated with TPA and a SerpinF2inhibitor (p=0.005). Treatment with TPA and a SerpinF2 inhibitor reducedneuronal cell death by comparison to TPA alone (FIG. 7A, p<0.01).Treatment with TPA and a SerpinF2 inhibitor also prevented hemorrhage bycomparison to TPA alone (FIG. 7B, p<0.001). Finally, the combination ofTPA and a SerpinF2 inhibitor significantly reduced hemispheric swellingby comparison to TPA alone (FIG. 7C, p<0.05). Taken together, thesestudies show that SerpinF2 inhibition reverses the effects of endogenousand exogenous TPA and significantly increases survival after ischemicstroke. This appears to be related to the fact that Serpin F inhibitionprevents hemorrhage and brain swelling which are major causes ofmortality and disability after stroke.

Since inhibition of SF2 reduces TPA-induced hemorrhage it may alsopreserve the integrity of the blood brain barrier in TPA-treated mice.In mice treated with TPA alone, there was leakage of albumin outsidevascular spaces identified by collagen IV immunostaining (FIG. 8A). Incontrast, albumin leakage was markedly reduced in mice treated with TPAand the SF2-I (FIG. 8A, p<0.05), consistent with reduced breakdown ofthe blood brain barrier. Matrix metalloproteinase-9 has been identifiedas a key mediator in breakdown of the blood brain barrier, hemorrhageand brain edema after TPA therapy.^(60,61) TPA-treated mice showedsignificantly greater expression of MMP-9 in the brain than control,untreated mice (p<0.01). Combination treatment with TPA and the SF2-Imarkedly reduced MMP-9 levels (FIG. 8B, p<0.01). TPA treatment alsosignificantly enhanced TUNEL staining, consistent with enhancedapoptosis in the stroke region (FIG. 8C). By comparison, the combinationof TPA and the SF2-I markedly reduced TUNEL staining (FIGS. 8C,p<0.001), consistent with protection against apoptosis.

In mechanical occlusion and brain injury models TPA expression isenhanced after brain injury.²⁸⁻³⁰ In these models, both endogenous andpharmacologic TPA are neurotoxic and SerpinF2 inhibitor reducesneurotoxicity.³¹⁻³³ Many different mechanisms have been proposed toexplain TPA's neurotoxocity.^(27,61) However, since the vast majority ofhuman strokes are due to thrombotic or thromboembolic arterialocclusion, it has been argued that the neurotoxicity of TPA observedwith non-thrombotic methods may have limited translational relevance tohuman ischemic stroke⁶² where the actions of TPA in dissolving thrombimay be neuroprotective. To examine the overall neuroprotective andneurotoxic effects of TPA in a manner that has translational relevanceto human stroke, the thromboembolic stroke model described by Zhang etal.⁴⁷ was modified. The result was a reproducible model of large vessel(MCA) thromboembolism that permits the simultaneous examination ofneuronal cell death, hemorrhage, fibrinolysis and swelling afterdifferent periods of ischemia.

Most humans present with stroke after 2 or more hours of ischemia. WhenTPA treatment was given 2.5 hrs. after thromboembolism, i.e., at timesthat more closely simulate the timing of human therapy, it hadneurotoxic effects. Despite successfully increasing the dissolution ofthe thromboemboli, TPA also significantly increased neuronal cell deathand cerebral hemorrhage. Treatment with TPA also affected survival afterthromboembolic stroke. Mice treated with TPA had significant mortality24 hours after treatment (78%). These lethal, neurotoxic effectsoccurred despite clear evidence that TPA was inducing systemicplasminogen activation as indicated by plasminogen (p<0.01) andfibrinogen consumption (p<0.001).

Previous studies with mechanical occlusion indicate that SerpinF2protects against the neurotoxicity of TPA.³⁶ Previous studies withSerpinF2 inhibitors show that they directly enhance TPA activity (U.S.Pat. No. 6,114,506). Increased TPA activity is associated with increasedneuronal cell death, hemorrhage (FIG. 5), death (FIG. 3), breakdown ofthe blood brain barrier, increased MMP-9 expression and apoptosis (FIG.6) after prolonged ischemia. Therefore, it is not expected that SerpinF2inhibitors would markedly reduce these neurotoxic effects of TPA.

In summary, in a thromboembolic model of ischemic stroke, standard andlow dose TPA caused neuronal cell death, with or without successfulfibrinolysis, after prolonged ischemia. In contrast to previouspredictions, treatment with an inhibitor of SerpinF2 markedly reducedthe neurotoxicity of pharmacologic and endogenous TPA and enhancedsurvival after thromboembolic stroke.

REFERENCES

-   1. Dohmen C, Galldiks N, Bosche B, Kracht L, Graf R. The severity of    ischemia determines and predicts malignant brain edema in patients    with large middle cerebral artery infarction. Cerebrovasc Dis 2012;    33:1-7.-   2. Westermaier T M, Stetter C M, Raslan M, Vince G H M P, Ernestus R    I M P. Brain edema formation correlates with perfusion deficit    during the first six hours after experimental subarachnoid    hemorrhage in rats. Exp Transl Stroke Med 2012; 4:8.-   3. Martinet V, Guigui B, Glacet-Bernard A, et al. Macular edema in    central retinal vein occlusion: correlation between optical    coherence tomography, angiography and visual acuity. Int Ophthalmol    2012.-   4. Abdel-Aty H, Cocker M, Meek C, Tyberg JV, Friedrich M G. Edema as    a very early marker for acute myocardial ischemia: a cardiovascular    magnetic resonance study. J Am Coll Cardiol 2009; 53:1194-201.-   5. Garcia-Dorado D, Andres-Villarreal M, Ruiz-Meana M, Inserte J,    Barba I. Myocardial edema: a translational view. J Mol Cell Cardiol    2012; 52:931-9.-   6. Chen K H, Chao D, Liu C F, Chen C F, Wang D. Ischemia and    reperfusion of the lung tissues induced increase of lung    permeability and lung edema is attenuated by dimethylthiourea    (PP69). Transplant Proc 2010; 42:748-50.-   7. Greca F H, Goncalves N M, Souza Filho Z A, Noronha L, Silva R F,    Rubin M R. The protective effect of methylene blue in lungs, small    bowel and kidney after intestinal ischemia and reperfusion. Acta Cir    Bras 2008; 23:149-56.-   8. Lee H T, Park S W, Kim M, D'Agati V D. Acute kidney injury after    hepatic ischemia and reperfusion injury in mice. Lab Invest 2009;    89:196-208.-   9. Fujimoto K, Hosotani R, Wada M, et al. Ischemia-reperfusion    injury on the pancreas in rats: identification of acinar cell    apoptosis. J Surg Res 1997; 71:127-36.-   10. Sage E, Mercier O, Van den Eyden F, et al. Endothelial cell    apoptosis in chronically obstructed and reperfused pulmonary artery.    Respir Res 2008; 9:19.-   11. Bekkers S C, Yazdani S K, Virmani R, Waltenberger J.    Microvascular obstruction: underlying pathophysiology and clinical    diagnosis. J Am Coll Cardiol 2010; 55:1649-60.-   12. Strong K, Mathers C, Bonita R. Preventing stroke: saving lives    around the world. Lancet neurology 2007; 6:182-7.-   13. Albers G W, Amarenco P, Easton J D, Sacco R L, Teal P.    Antithrombotic and thrombolytic therapy for ischemic stroke:    American College of Chest Physicians Evidence-Based Clinical    Practice Guidelines (8th Edition). Chest 2008; 133:630S-69S.-   14. Fieschi C, Argentino C, Lenzi G L, Sacchetti M L, Toni D,    Bozzao L. Clinical and instrumental evaluation of patients with    ischemic stroke within the first six hours. Journal of the    neurological sciences 1989; 91:311-21.-   15. Viitanen M, Winblad B, Asplund K. Autopsy-verified causes of    death after stroke. Acta Med Scand 1987; 222:401-8.-   16. Bamford J, Dennis M, Sandercock P, Burn J, Warlow C. The    frequency, causes and timing of death within 30 days of a first    stroke: the Oxfordshire Community Stroke Project. J Neurol Neurosurg    Psychiatry 1990; 53:824-9.-   17. Braga P, Ibarra A, Rega I, et al. Prediction of early mortality    after acute stroke. J Stroke Cerebrovasc Dis 2002; 11:15-22.-   18. Silver F L, Norris J W, Lewis A J, Hachinski V C. Early    mortality following stroke: a prospective review. Stroke 1984;    15:492-6.-   19. Koennecke H C, Belz W, Berfelde D, et al. Factors influencing    in-hospital mortality and morbidity in patients treated on a stroke    unit. Neurology 2011; 77:965-72.-   20. Johnston K C, Wagner D P, Wang X Q, et al. Validation of an    acute ischemic stroke model: does diffusion-weighted imaging lesion    volume offer a clinically significant improvement in prediction of    outcome? Stroke 2007; 38:1820-5.-   21. Saver J L, Johnston K C, Homer D, et al. Infarct volume as a    surrogate or auxiliary outcome measure in ischemic stroke clinical    trials. The RANTTAS Investigators. Stroke 1999; 30:293-8.-   22. Marler J R, Goldstein L B. Medicine. Stroke—tPA and the clinic.    Science 2003; 301:1677.-   23. Alexandrov A V, Demchuk A M, Felberg R A, et al. High rate of    complete recanalization and dramatic clinical recovery during tPA    infusion when continuously monitored with 2-MHz transcranial doppler    monitoring. Stroke 2000; 31:610-4.-   24. Kwiatkowski T G, Libman R B, Frankel M, et al. Effects of tissue    plasminogen activator for acute ischemic stroke at one year.    National Institute of Neurological Disorders and Stroke Recombinant    Tissue Plasminogen Activator Stroke Study Group. N Engl J Med 1999;    340:1781-7.-   25. Levy D E, Brott T G, Haley E C, Jr., et al. Factors related to    intracranial hematoma formation in patients receiving tissue-type    plasminogen activator for acute ischemic stroke. Stroke 1994;    25:291-7.-   26. Lees K R, Bluhmki E, von Kummer R, et al. Time to treatment with    intravenous alteplase and outcome in stroke: an updated pooled    analysis of ECASS, ATLANTIS, NINDS, and EPITHET trials. Lancet 2010;    375:1695-703.-   27. Vivien D, Buisson A. Serine protease inhibitors: novel    therapeutic targets for stroke? J Cereb Blood Flow Metab 2000;    20:755-64.-   28. Stehling F, Weber R, Ozcelik A, et al. Acute changes of    coagulation and fibrinolysis parameters after experimental    thromboembolic stroke and thrombolytic therapy. Neuroscience letters    2008; 441:39-43.-   29. Dietzmann K, von Bossanyi P, Krause D, Wittig H, Mawrin C,    Kirches E. Expression of the plasminogen activator system and the    inhibitors PAI-1 and PAI-2 in posttraumatic lesions of the CNS and    brain injuries following dramatic circulatory arrests: an    immunohistochemical study. Pathology, research and practice 2000;    196:15-21.-   30. Nagai N, Suzuki Y, Van Hoef B, Lijnen H R, Collen D. Effects of    plasminogen activator inhibitor-1 on ischemic brain injury in    permanent and thrombotic middle cerebral artery occlusion models in    mice. J Thromb Haemost 2005; 3:1379-84.-   31. Wang Y F, Tsirka S E, Strickland S, Stieg P E, Soriano S G,    Lipton S A. Tissue plasminogen activator (tPA) increases neuronal    damage after focal cerebral ischemia in wild-type and tPA-deficient    mice. Nat Med 1998; 4:228-31.-   32. Nagai N, De Mol M, Lijnen H R, Carmeliet P, Collen D. Role of    plasminogen system components in focal cerebral ischemic infarction:    a gene targeting and gene transfer study in mice. Circulation 1999;    99:2440-4.-   33. Sheehan J J, Tsirka S E. Fibrin-modifying serine proteases    thrombin, tPA, and plasmin in ischemic stroke: a review. Glia 2005;    50:340-50.-   34. Tsirka S E, Gualandris A, Amaral D G, Strickland S.    Excitotoxin-induced neuronal degeneration and seizure are mediated    by tissue plasminogen activator. Nature 1995; 377:340-4.-   35. Tsirka S E, Bugge T H, Degen J L, Strickland S. Neuronal death    in the central nervous system demonstrates a non-fibrin substrate    for plasmin. Proceedings of the National Academy of Sciences of the    United States of America 1997; 94:9779-81.-   36. Tsirka S E, Rogove A D, Bugge T H, Degen J L, Strickland S. An    extracellular proteolytic cascade promotes neuronal degeneration in    the mouse hippocampus. J Neurosci 1997; 17:543-52.-   37. Yepes M, Sandkvist M, Coleman T A, et al. Regulation of seizure    spreading by neuroserpin and tissue-type plasminogen activator is    plasminogen-independent. J Clin Invest 2002; 109:1571-8.-   38. Suri M F, Yamagishi K, Aleksic N, Hannan P J, Folsom A R. Novel    hemostatic factor levels and risk of ischemic stroke: the    Atherosclerosis Risk in Communities (ARIC) Study. Cerebrovasc Dis    2010; 29:497-502.-   39. Marti-Fabregas J, Borrell M, Cocho D, et al. Hemostatic markers    of recanalization in patients with ischemic stroke treated with    rt-PA. Neurology 2005; 65:366-70.-   40. Roelofs J J, Rouschop K M, Leemans J C, et al. Tissue-type    plasminogen activator modulates inflammatory responses and renal    function in ischemia reperfusion injury. J Am Soc Nephrol 2006;    17:131-40.-   41. Zhao Y, Sharma A K, LaPar D J, et al. Depletion of tissue    plasminogen activator attenuates lung ischemia-reperfusion injury    via inhibition of neutrophil extravasation. Am J Physiol Lung Cell    Mol Physiol 2011; 300:L718-29.-   42. Hong T T, Huang J, Lucchesi B R. Effect of thrombolysis on    myocardial injury: recombinant tissue plasminogen activator vs.    alfimeprase. Am J Physiol Heart Circ Physiol 2006; 290:H959-67.-   43. Kumada M, Niwa M, Wang X, et al. Endogenous tissue type    plasminogen activator facilitates NMDA-induced retinal damage.    Toxicol Appl Pharmacol 2004; 200:48-53.-   44. Del Zoppo G J. Focal cerebral ischemia and hemostasis: a PAI-1    conundrum. J Thromb Haemost 2005; 3:1376-8.-   45. Aoki T, Sumii T, Mori T, Wang X, Lo E H. Blood-brain barrier    disruption and matrix metalloproteinase-9 expression during    reperfusion injury: mechanical versus embolic focal ischemia in    spontaneously hypertensive rats. Stroke 2002; 33:2711-7.-   46. Asahi M, Huang Z, Thomas S, et al. Protective effects of statins    involving both eNOS and tPA in focal cerebral ischemia. J Cereb    Blood Flow Metab 2005; 25:722-9.-   47. Zhang Z, Chopp M, Zhang R L, Goussev A. A mouse model of embolic    focal cerebral ischemia. J Cereb Blood Flow Metab 1997; 17:1081-8.-   48. Zhang Z G, Zhang L, Ding G, et al. A model of mini-embolic    stroke offers measurements of the neurovascular unit response in the    living mouse. Stroke 2005; 36:2701-4.-   49. Houng A K, McNamee R A, Kerner A, et al. Atrial natriuretic    peptide increases inflammation, infarct size, and mortality after    experimental coronary occlusion. American journal of physiology    2009; 296:H655-61.-   50. Swanson R A, Morton M T, Tsao-Wu G, Savalos R A, Davidson C,    Sharp F R. A semiautomated method for measuring brain infarct    volume. J Cereb Blood Flow Metab 1990; 10:290-3.-   51. Robinson B R, Houng A K, Reed G L. Catalytic life of activated    factor XIII in thrombi. Implications for fibrinolytic resistance and    thrombus aging. Circulation 2000; 102:1151-7.-   52. Sazonova I Y, McNamee R A, Houng A K, King S M, Hedstrom L, Reed    G L. Reprogrammed streptokinases develop fibrin-targeting and    dissolve blood clots with more potency than tissue plasminogen    activator. J Thromb Haemost 2009; 7:1321-8.-   53. Zunker P, Schick A, Padro T, Kienast J, Phillips A, Ringelstein    E B. Tissue plasminogen activator and plasminogen activator    inhibitor in patients with acute ischemic stroke: relation to stroke    etiology. Neurological research 1999; 21:727-32.-   54. Wang X, Lee S R, Arai K, et al. Lipoprotein receptor-mediated    induction of matrix metalloproteinase by tissue plasminogen    activator. Nat Med 2003; 9:1313-7.-   55. Hunter A J, Hatcher J, Virley D, et al. Functional assessments    in mice and rats after focal stroke. Neuropharmacology 2000;    39:806-16.-   56. Heinsius T, Bogousslaysky J, Van Melle G. Large infarcts in the    middle cerebral artery territory. Etiology and outcome patterns.    Neurology 1998; 50:341-50.-   57. Yepes M, Sandkvist M, Moore E G, Bugge T H, Strickland D K,    Lawrence D A. Tissue-type plasminogen activator induces opening of    the blood-brain barrier via the LDL receptor-related protein. J Clin    Invest 2003; 112:1533-40.-   58. Asahi M, Wang X, Mori T, et al. Effects of matrix    metalloproteinase-9 gene knock-out on the proteolysis of blood-brain    barrier and white matter components after cerebral ischemia. J    Neurosci 2001; 21:7724-32.-   59. Montaner J, Molina C A, Monasterio J, et al. Matrix    metalloproteinase-9 pretreatment level predicts intracranial    hemorrhagic complications after thrombolysis in human stroke.    Circulation 2003; 107:598-603.-   60. Wang X, Tsuji K, Lee S R, et al. Mechanisms of hemorrhagic    transformation after tissue plasminogen activator reperfusion    therapy for ischemic stroke. Stroke 2004; 35:2726-30.-   61. Kaur J, Zhao Z, Klein G M, Lo E H, Buchan A M. The neurotoxicity    of tissue plasminogen activator? J Cereb Blood Flow Metab 2004;    24:945-63.-   62. Tabrizi P, Wang L, Seeds N, et al. Tissue plasminogen activator    (tPA) deficiency exacerbates cerebrovascular fibrin deposition and    brain injury in a murine stroke model: studies in tPA-deficient mice    and wild-type mice on a matched genetic background.    Arteriosclerosis, thrombosis, and vascular biology 1999; 19:2801-6.

1-28. (canceled)
 29. A method of treating or preventing hemorrhage oredema in a patient in need thereof, comprising administering to thepatient an effective amount of an anti-SerpinF2 antibody or fragmentthereof that reduces SerpinF2 activity or concentration, wherein thehemorrhage or edema is cardiac, hepatic, pancreatic, respiratory orrenal.
 30. The method of claim 29, wherein anti-SerpinF2 antibody orfragment thereof is administered in a dose range from 28-91nanomoles/kg.
 31. The method of claim 29, wherein the anti-SerpinF2antibody or fragment thereof is administered in a dose range from 17-50nanomoles/kg.
 32. The method of claim 29, wherein the anti-SerpinF2antibody or fragment thereof is administered in a dose range from0.5-1.0 moles inhibitor per mole of SerpinF2.
 33. A method of inhibitingdisability or death from endogenous or pharmacologic tissue plasminogenactivator (TPA) mediated hemorrhage or edema in a patient in needthereof, the method comprising administering to the patient an effectiveamount of an anti-SerpinF2 antibody or fragment thereof that reducesSerpinF2 activity or concentration, wherein the hemorrhage or edema iscardiac, hepatic, pancreatic, respiratory or renal.
 34. The method ofclaim 33, further comprising determining that the patient is at risk forTPA induced damage.
 35. The method of claim 34, wherein the damagecomprises cardiac, hepatic, pancreatic, respiratory or renal damage. 36.The method of claim 35, wherein the determining step further comprisesdetermining that the patient has cardiac, hepatic, pancreatic,respiratory or renal symptoms indicative of cardiac, hepatic,pancreatic, respiratory or renal damage.
 37. The method of claim 33,wherein the method treats or prevents TPA toxicity.
 38. The method ofclaim 37, wherein the TPA toxicity is due to trauma.
 39. The method ofclaim 33, wherein a plasminogen activator or serine protease enzyme hasbeen previously administered to the patient within 48 hours.
 40. Themethod of claim 39, wherein TPA has been previously administered to thepatient within 48 hours.
 41. The method of claim 33, whereinanti-SerpinF2 antibody or fragment thereof is administered in a doserange from 28-91 nanomoles/kg.
 42. The method of claim 33, wherein theanti-SerpinF2 antibody or fragment thereof is administered in a doserange from 17-50 nanomoles/kg.
 43. The method of claim 33, wherein theanti-SerpinF2 antibody or fragment thereof is administered in a doserange from 0.5-1.0 moles inhibitor per mole of SerpinF2.
 44. A method ofinhibiting edema or tissue plasminogen activator (TPA) toxicity in alung of a patient in need thereof comprising administering to saidpatient an effective amount of an anti-SerpinF2 antibody or fragmentthereof that reduces SerpinF2 concentration or activity in said patientto inhibit the organ edema or tissue plasminogen activator (TPA)toxicity.
 45. The method of claim 44, wherein TPA has been previouslyadministered to the patient within 48 hours.
 46. The method of claim 44,wherein the SerpinF2 binding inhibitor in an antibody and isadministered in a dose range from 28-91 nanomoles/kg.
 47. The method ofclaim 44, wherein the anti-SerpinF2 antibody or fragment thereof isadministered in a dose range from 17-50 nanomoles/kg.
 48. The method ofclaim 44, wherein the anti-SerpinF2 antibody or fragment thereof isadministered in a dose range from 0.5-1.0 moles inhibitor per mole ofSerpinF2.
 49. A method of treating or preventing morbidity ordisability, or preventing death, from cellular damage, hemorrhage, ororgan swelling after tissue injury, due to SerpinF2 activity, in apatient in need thereof, the method comprising administering to thepatient an effective amount of an anti-SerpinF2 antibody or fragmentthereof that reduces SerpinF2 activity or concentration, wherein thecellular damage, hemorrhage, or organ swelling is cardiac, hepatic,pancreatic, respiratory or renal.
 50. The method of claim 49, whereinthe SerpinF2 activity is present in conditions associated with increasedlevels of at least one plasminogen activator.
 51. The method of claim50, wherein the plasminogen activator or a serine protease enzyme hasbeen previously administered to the patient within 48 hours.
 52. Themethod of claim 51, wherein the at least one plasminogen activatorcomprises tissue plasminogen activator (TPA).
 53. The method of claim49, further comprising determining that the patient has cardiac,hepatic, pancreatic, respiratory or renal symptoms indicative ofcardiac, hepatic, pancreatic, respiratory or renal cellular damage,hemorrhage, or organ swelling.
 54. The method of claim 49, whereinanti-SerpinF2 antibody or fragment thereof is administered in a doserange from 28-91 nanomoles/kg.
 55. The method of claim 49, wherein theanti-SerpinF2 antibody or fragment thereof is administered in a doserange from 17-50 nanomoles/kg.
 56. The method of claim 49, wherein theanti-SerpinF2 antibody or fragment thereof is administered in a doserange from 0.5-1.0 moles inhibitor per mole of SerpinF2.